CAR T-Cell Characterization

CAR T-Cell Therapy Characterization

Selecting CAR T-cell therapy targets is a critical development step. To be effective, CAR T cells need to target specific receptors on tumor cells. These receptors can vary based on cancer type, which complicates CAR T-cell therapy development.

Because of the intricacies of these treatments, CAR T-cell therapy development is multifaceted, requiring a variety of tools to assess them throughout the process. ​

CAR T-Cell Phenotype Analysis

Harnessing the power of the immune system with therapeutic CAR T cells requires deep investigation of complex cellular populations. As investigators learn more about these therapeutics, deeper characterization of additional cellular targets is required.​

The speed of flow cytometry and its ability to scrutinize complex phenotypes at the single-cell level gives researchers the power to rapidly analyze and characterize millions of cells in a matter of seconds even with a large number of parameters. Furthermore, these multiplex, large-scale assays can benefit from automated high-throughput, high-parameter flow cytometry in order to phenotype cells more quickly.​​

In addition to Bio-Rad’s flow cytometry instrumentation, we offer off the shelf as well as custom antibodies to support the entire research and development process, from target identification to quality control during clinical trials.​

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Early Identification of CAR T-Cell Therapy Targets

Characterizing the cell types and therapeutic targets relevant to the activity of CAR T cells is of paramount importance for their creation. High-throughput, high-parameter flow cytometry aids in characterizing these entities with accuracy and speed.

Use Bio-Rad’s flow cytometry solutions in early CAR T-cell research to:

  • Evaluate editing efficiencies of a specific CAR
  • Detect CAR during isolation and activation and ensure other indicators of functionality
  • Identify relevant receptors on tumor cells and T cells to target for modification

Our ZE5 Cell Analyzer is a high performance, automation-ready flow cytometer with the ideal combination of high-throughput screening and high parameter cell analysis for the evaluation of CAR T-cell therapy targets.

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Differentiating CAR T Cells with Custom Antibodies​

Confirming CAR T-cell transduction or transfection and differentiating between CAR T cells and the patient’s normal T cells requires a highly specific antibody against the scFv of the CAR. This is necessary for use in flow cytometry and required throughout preclinical and clinical development.

Bio-Rad's custom antibody team are experts in the generation of recombinant anti-idiotypic antibodies, a highly specialized, critical reagent needed for analyzing CAR T-cell transduction or transfection and cellular kinetics​

Bio-Rad's custom service offers:

  • Antibody specificity against the unique determinants of the CAR
  • Multiple format and conjugation options from the outset using SpyTag technology
  • Antibodies dispatched in less than 3 months
  • Recombinant production — reliable, consistent quality, easy scale-up
  • Antibody sequence available for security and independence

Talk to our antibody experts to discover how you can get the exact antibodies you need to support your CAR T-cell transfection studies and complete therapy development.

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Confidently Probe for CAR Expression​

In the early development of CAR T-cell therapies, it can be challenging to measure and confirm CAR expression levels with confidence. This is particularly difficult if you are relying on housekeeping proteins as your loading control, which have been shown to have limited dynamic range and reliability in comparison to total protein normalization.​

Bio-Rad’s Stain-Free Western Blotting can help by using total protein normalization with Stain-Free technology. This leads not only to more reliable data but can also produce results more quickly compared to traditional western blotting protocols.

  • Visualize gels minutes after electrophoresis
  • Perform quality assessments at each step without the need for additional staining and destaining
  • Increase accuracy and produce reliable quantitative results across a larger dynamic range.

Stain-Free Western Blotting allows for normalization across a wider dynamic range, giving you the confidence to select the best CAR gene in the development of your CAR T-cell therapy.​

Stain-Free Total Protein Normalization for Western Blotting

Linearity comparison of Stain-Free total protein measurement and immunodetection of three housekeeping proteins in 10–50 μg of HeLa cell lysate. On the left are representative images of (a), Stain-Free blot and the chemiblots for (b), β-actin; (c), β-tubulin and (d), GAPDH. Lane labels correspond to total protein load (μg). Although the actin and tubulin signals appear linear, the densitometric ratio was far below the predicted “quantitative response” of actual loading whereas the Stain-Free signal correlated to the expected result (e).

Reference
Taylor SC Posch A (2014). The design of a quantitative western blot experiment. Biomed Res Int. 36, 1590

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